DNA vaccines encoding heat shock proteins

ABSTRACT

The present invention is related to recombinant constructs encoding heat shock proteins or active fragments thereof that are effective in treating T cell mediated inflammatory autoimmune diseases by DNA vaccines. The treatment with the recombinant constructs of the present invention provides long-term expression of specific heat shock proteins for fragments thereof. In one embodiment, the present invention is related to a recombinant construct of a nucleic acid sequence encoding HSP60, HSP70 or HSP90. The present invention also is related to a recombinant construct of a nucleic acid sequence selected from amino acids 1-140 of HSP60, amino acids 130-260 of HSP60 and amino acids 31-50 of HSP60. Another aspect of the present invention is related to an active fragment of HSP60 corresponding to amino acids 31-50 of HSP60 effective in treating arthritis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International applicationPCT/IL03/00417 filed May 21, 2003, which claims priority to U.S.provisional patent application No. 60/381,821 filed May 21, 2002, theentire content of which is expressly incorporated herein by referencethereto.

FIELD OF THE INVENTION

The present invention relates to recombinant constructs encoding heatshock proteins or active fragments thereof, effective in treating T cellmediated diseases including inflammatory autoimmune diseases by DNAvaccination. The present invention further relates to compositions andmethods for treating T cell mediated diseases.

BACKGROUND OF THE INVENTION

While the normal immune system is closely regulated, aberrations inimmune responses are not uncommon. In some instances, the immune systemfunctions inappropriately and reacts to a component of the host as if itwere, in fact, foreign. Such a response results in an autoimmunedisease, in which the host's immune system attacks the host's owntissue. T cells, as the primary regulators of the immune system,directly or indirectly effect such autoimmune pathologies. Tcell-mediated autoimmune diseases refer to any condition in which aninappropriate T cell response is a component of the disease. Thisincludes both diseases directly mediated by T cells, and also diseasesin which an inappropriate T cell response contributes to the productionof abnormal antibodies.

Numerous diseases are believed to result from autoimmune mechanisms.

Prominent among these are rheumatoid arthritis, systemic lupuserythematosus, multiple sclerosis, Type I diabetes, myasthenia gravisand pemphigus vulgaris. AUTOIMMUNE diseases affect millions ofindividuals world-wide and the cost of these diseases, in terms ofactual treatment expenditures and lost productivity, is measured inbillions of dollars annually.

Adjuvant arthritis (AA) is an experimental autoimmune disease thatmodels several features of human rheumatoid arthritis (1). AA is inducedin Lewis rats by immunization with heat killed Mycobacteriumtuberculosis (MT) suspended in Incomplete Freund's Adjuvant (IFA) (1).T-cell reactivity against the mycobacterial 65 kDa heat shock protein(HSP65) is involved in the progression of AA. HSP65-specific T-cellsdirected against an epitope formed by aa 180-188 (2) cross-react with aself-antigen present in cartilage (3) and can adoptively transfer AA(4,5). However, vaccination with HSP65 or HSP65-peptides can alsoprevent the development of AA (6-11). The regulatory properties of HSP65in AA are thought to involve the activation of T-cells cross-reactivewith the endogenous 60 kDa heat shock protein (HSP60) (12). Thishypothesis is supported by the finding that immunization with arecombinant vaccinia virus encoding human HSP60 (about 95% homologous torat HSP60) prevents (13) or treats (14) AA. The inventor of the presentinvention have recently reported that DNA vaccination with human HSP60prevents AA (15). Protection from AA was associated with the activationof T-cells responding to HSP60 (15). The human hsp60 molecule wasformerly designated HSP65, but is now designated HSP60 in view of moreaccurate molecular weight information; by either designation, theprotein is the same.

A preferable method for treating autoimmune diseases includes modulatingthe immune system of a patient to assist the patient's natural defensemechanisms. Traditional reagents and methods used to attempt to regulatean immune response in a patient also result in unwanted side effects andhave limited effectiveness. For example, immunosuppressive reagents(e.g., cyclosporin A, azathioprine, and prednisone) used to treatpatients with autoimmune diseases also suppress the patient's entireimmune response, thereby increasing the risk of infection. In addition,immunopharmacological reagents used to treat cancer (e.g., interleukins)are short-lived in the circulation of a patient and are ineffectiveexcept in large doses. Due to the medical importance of immuneregulation and the inadequacies of existing immunopharmacologicalreagents, reagents and methods to regulate specific parts of the immunesystem have been the subject of study for many years.

EP 262710 of Cohen et al. discloses the use of HSP65, or fragmentsthereof for the preparation of compositions for the alleviation,treatment and diagnosis of autoimmune diseases, especially arthriticconditions. EP 322990 of Cohen et al. discloses that a polypeptidehaving amino acid sequence 172-192 of HSP65 is capable of inducingresistance to auto-immune arthritis and similar auto-immune diseases. WO92/04049 of Boog et al. discloses peptides derived from Mycobacteriumtuberculosis protein HSP-65 containing at least 7 amino acid residuesand inhibits antigen recognition by T lymphocytes in treatment ofarthritis and organ rejection.

WO 01/57056 of Karin discloses a method of treating rheumatoidarthritis. The method comprising the step of expressing within theindividual at least an immunologically recognizable portion of acytokine from an exogenous polynucleotide encoding at least a portion ofthe cytokine, wherein a level of expression of the at least a portion ofthe cytokine is sufficient to induce the formation of anti-cytokineimmunoglobulins which serve for neutralizing or ameliorating theactivity of a respective and/or cross reactive endogenous cytokine, tothereby treat rheumatoid arthritis. U.S. Pat. No. 6,316,420 to Karin andcoworkers further discloses DNA cytokine vaccines and use of same forprotective immunity against multiple sclerosis.

WO 02/16549 of Cohen et al., assigned to the assignee of the presentinvention, relates to DNA vaccines useful for the prevention andtreatment of ongoing autoimmune diseases. The compositions and methodsof the invention feature the CpG oligonucleotide, preferably in a motifflanked by two 5′ purines and two 3′ pyrimidines. The vaccinesoptionally further comprise DNA encoding a peptide or a polypeptideselected from the group consisting of Hsp60, p277 or p277 variants. Thatdisclosure is directed to methods and compositions for the ameliorativetreatment of ongoing autoimmune disease in general and Insulin DependentDiabetes Mellitus (IDDM) in particular.

U.S. Pat. No. 5,993,803 discloses that when HSP60, or peptides andanalogs thereof, are administered in a recipient subject beforetransplantation of an organ or tissue, autoimmunity to HSP60 isdown-regulated, resulting in the prevention or suppression of graftrejection of the transplanted organ or tissue.

WO 00/27870 of Naparstek and colleagues discloses a series of relatedpeptides derived from heat shock proteins HSP65 and HSP60, theirsequences, antibodies, and use as vaccines for conferring immunityagainst autoimmune and/or inflammatory disorders such as arthritis.These peptides are intended by the inventors to represent the shortestsequence or epitope that is involved in protection of susceptible ratstrains against adjuvant induced arthritis. These sequences furtherdisclose what the inventors identify as the common “protective motif”.

There exists a long-felt need for an effective means of curing orameliorating T cell mediated inflammatory autoimmune diseases. None ofthe background art discloses DNA vaccines encoding heat shock proteinsfor treating T cell mediated inflammatory autoimmune diseases. Such atreatment should ideally control the inappropriate T cell response,rather than merely reducing the symptoms.

SUMMARY OF THE INVENTION

DNA vaccination represents a novel and unexpectedly effective means ofexpressing antigen in vivo for the generation of both humoral andcellular immune responses. The present invention uses this technology toelicit protective immunity against T cell-mediated autoimmune diseases.The compositions and methods of the present invention are effective inany T-cell mediated inflammatory autoimmune disease including but notlimited to: rheumatoid arthritis, collagen II arthritis, multiplesclerosis, autoimmune neuritis, systemic lupus erythematosus, psoriasis,juvenile onset diabetes, Sjogren's disease, thyroid disease,sarcoidosis, autoimmune uveitis, inflammatory bowel disease (Crohn's andulcerative colitis) or autoimmune hepatitis.

In one aspect, the present invention is related to DNA vaccines encodingheat shock proteins for treating T cell-mediated inflammatory autoimmunediseases. According to various specific embodiments of the presentinvention, the heat shock proteins are mammalian heat shock proteins,preferably the full-length heat shock protein 60 (HSP60), thefull-length heat shock protein 70 (HSP70) or the full-length heat shockprotein 90 (HSP90). The heat shock proteins according to the presentinvention are preferably human heat shock proteins, however othermammalian heat shock proteins are within the scope of the presentinvention.

According to various additional embodiments of the present invention,the DNA vaccines encode active fragments of HSP60, HSP70 or HSP90. Incertain specific embodiments, the DNA vaccines encode active fragmentsof HSP60. Preferred fragments of HSP60 correspond to amino acids 1-140of HSP60 (SEQ ID NO:1), amino acids 130-260 of HSP60 (SEQ ID NO:2) oramino acids 31-50 of HSP60 (SEQ ID NO:3).

The treatment with the DNA vaccines of the present invention provideslong-term expression of specific heat shock proteins or active fragmentsthereof. Such long-term expression allows for the maintenance of aneffective, but non-toxic, dose of the encoded polypeptides to treat thedisease and limits the frequency of administration of the therapeuticcomposition needed to treat an animal. In addition, because of the lackof toxicity, therapeutic compositions of the present invention can beused in repeated treatments.

In another aspect, the present invention is related to novel recombinantconstructs comprising a nucleic acid sequence encoding at least part ofa heat shock protein being operatively linked to at least onetranscription control element. According to various embodiments of thepresent invention, the heat shock protein is a mammalian heat shockprotein, preferably the full-length HSP60, the full-length HSP70 or thefull-length HSP90. The heat shock proteins according to the presentinvention are preferably human heat shock proteins, however othermammalian heat shock proteins are within the scope of the presentinvention.

According to various embodiments of the present invention, therecombinant constructs encoding active fragments of HSP60, HSP70 orHSP90. In a preferred embodiment, the recombinant constructs encodeactive fragments of HSP60, said active fragments selected from: aminoacids 1-140 of HSP60 (SEQ ID NO:1), amino acids 130-260 of HSP60 (SEQ IDNO:2) or amino acids 31-50 of HSP60 (SEQ ID NO:3), the nucleic acidsequence being operatively linked to at least one transcription controlelement.

According to various specific embodiments, the constructs of the presentinvention comprise at least one transcription control element selectedfrom the group consisting of: RSV control sequences, CMV controlsequences, retroviral LTR sequences, SV-40 control sequences and β-actincontrol sequences.

In another aspect, the present invention is related to an eukaryoticexpression vector comprising the recombinant constructs of the presentinvention. According to various embodiments, the eukaryotic expressionvector is selected from pcDNA3, pcDNA3.1(+/−), pZeoSV2(+/−), pSecTag2,pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pCI, pBK-RSV, pBK-CMV andpTRES.

Another aspect of the present invention provides a pharmaceuticalcomposition effective for treating a T cell-mediated inflammatoryautoimmune disease, the composition comprising (a) a recombinantconstruct comprising an isolated nucleic acid sequence encoding a heatshock protein, or an active fragment thereof, the nucleic acid sequencebeing operatively linked to one or more transcription control sequences;and (b) a pharmaceutically acceptable carrier.

In one embodiment, the nucleic acid sequence encodes the full-lengthHSP60, the full-length HSP70 or the full-length HSP90. In anotherembodiment, the nucleic acid sequence encoding an active fragment ofHSP60, HSP70 or HSP90. In a preferred embodiment, the nucleic acidsequence encoding amino acids 1-140 of human HSP60 (SEQ ID NO:1). Inanother preferred embodiment, the nucleic acid sequence encoding aminoacids 130-260 of human HSP60 (SEQ ID NO:2). In yet another preferredembodiment, the nucleic acid sequence encoding amino acids 31-50 ofhuman HSP60 (SEQ ID NO:3).

The pharmaceutical compositions comprising the recombinant constructsaccording to the present invention may advantageously compriseliposomes, micelles, emulsions or cells. Still further embodimentsutilize a virus as is known in the art in order to introduce and expressthe nucleic acid sequences according to the present invention in thehost cells.

In another aspect, the present invention is related to a method ofinhibiting or preventing the symptoms of a T-cell mediated inflammatoryautoimmune disease, the method comprising administering to an individualin need of said treatment, preferably a human individual, a therapeuticcomposition comprising a recombinant construct, said recombinantconstruct comprising an isolated nucleic acid sequence encoding a heatshock protein, or a fragment thereof, thereby inhibiting or preventingthe symptoms of said autoimmune disease.

In one embodiment, the nucleic acid sequence encodes the full-lengthHSP60, the full-length HSP70 or the full-length HSP90, the nucleic acidsequence being operatively linked to at least one transcription controlelement. In another embodiment, the nucleic acid sequence corresponds toan active fragment of HSP60, HSP70 or HSP90. In a preferred embodiment,the nucleic acid sequence corresponds to amino acids 1-140 of humanHSP60 designated herein as pI (SEQ ID NO:1). In another preferredembodiment, the nucleic acid sequence corresponds to amino acids 130-260of human HSP60, designated herein as pII (SEQ ID NO:2). In yet anotherpreferred embodiment, the recombinant construct comprising a nucleicacid sequence corresponding to amino acids 31-50 of human HSP60 (SEQ IDNO:3).

According to various embodiments, the compositions and methods of thepresent invention are effective in any T-cell mediated inflammatoryautoimmune disease such as: rheumatoid arthritis, collagen II arthritis,multiple sclerosis, autoimmune neuritis, systemic lupus erythematosus,psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease,sarcoidosis, autoimmune uveitis, inflammatory bowel disease (Crohn's andulcerative colitis) or autoimmune hepatitis.

The present invention is particularly exemplified by the animal diseasemodel of adjuvant arthritis (AA), a T cell-mediated autoimmune diseasethat serves as an experimental model for rheumatoid arthritis. Thismodel is intended as a non-limitative example used for illustrativepurposes of the principles of the invention

In one embodiment, the therapeutic composition of the present inventionis administered to an individual at risk of developing a T-cell mediatedinflammatory autoimmune disease, thus serving as a preventive treatment.In another embodiment, the therapeutic composition of the presentinvention is administered to an individual during the initial stages ofthe disease or after the appearance of disease symptoms.

According to another aspect, the present invention provides a method fortreating a T cell-mediated inflammatory autoimmune disease comprisingthe steps of (a) obtaining cells from an individual; (b) transfectingthe cells ex vivo with a recombinant construct comprising an isolatednucleic acid sequence encoding a heat shock protein, or a fragmentthereof, the nucleic acid sequence being operatively linked to one ormore transcription control sequences; and (c) reintroducing thetransfected cells to the individual.

According to another aspect, the present invention provides a method fortreating a T cell-mediated inflammatory autoimmune disease comprisingthe steps of (a) obtaining cells from an individual; (b) infecting thecells ex vivo with a virus comprising a recombinant construct comprisingan isolated nucleic acid sequence encoding a heat shock protein, or afragment thereof, the nucleic acid sequence being operatively linked toone or more transcription control sequences; and (c) reintroducing theinfected cells to the individual.

According to another aspect, the present invention provides a method fortreating a T cell-mediated inflammatory autoimmune disease comprisingadministering to an individual in need thereof a therapeutic compositioncomprising (a) a fragment of mammalian HSP60 having amino acid sequenceselected from SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; and (b) apharmaceutically acceptable carrier.

According to another aspect, the present invention provides a method oftreating arthritis, said method comprising administering to anindividual in need thereof a therapeutic composition comprising (a) afragment of mammalian HSP60 having amino acid sequence Lys Phe Gly AlaAsp Ala Arg Ala Leu Met Leu Gln Gly Val Asp Leu Leu Ala Asp Alacorresponding to amino acid residus 31-50 of human HSP60 (denoted as SEQID NO:3); and (b) a pharmaceutically acceptable carrier, therebytreating arthritis. According to various embodiments, the carriercomprises a delivery vehicle that delivers the fragment to theindividual.

According to another aspect, the present invention provides a method oftreating arthritis, said method comprising the steps of (a) obtainingcells from an individual; (b) exposing the cells ex vivo with an activeamount of a fragment of mammalian HSP60 having amino acid sequencecorresponding to amino acids 31-50 of HSP60 (denoted as SEQ ID NO:3);and (c) reintroducing the exposed cells to the individual, therebytreating arthritis. In a preferred embodiment the cells are autologous Tcells. In another preferred embodiment, the mammalian HSP60 is humanHSP60.

These and further embodiments will be apparent from the detaileddescription and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Prevention of AA by vaccination with pI and pII. A. Time courseof AA. Rats were vaccinated in the quadriceps three times (on days-40,-26-12 relative to AA induction) with 150 μg of pcDNA3, pI, pII, pIII,pIV or pV. On day 0, AA was induced by injecting 1 mg of Mt suspended in100 μl of IFA, and arthritis scores were assessed every two or threedays starting at day 10. Bars show the mean±SEM assessment of diseaseseverity. B. Leg swelling measured at day 26 after AA induction.

FIG. 2: pHSP60 and pI-vaccination activate T-cell responses to Hu3. A.Proliferative responses (Stimulation Index, SI) to overlaping peptidescorresponding to the first 260 aa of HSP60. Rats vaccinated were killedand LNC were collected on day 26 after induction of AA. B. Dose-responseto Hu3 of pHSP60, pHSP65 and pcDNA3-vaccinated rats. Rats vaccinatedwere killed and LNC were collected on day 26 after induction of AA. C.Dose-response to Hu3 of pI, pII and pcDNA3-vaccinated rats. Ratsvaccinated were killed and LNC were collected on day 26 after inductionof AA.

FIG. 3: Prevention of AA by vaccination with Hu3. A. Time course of AA.Rats were vaccinated once (on day-7 relative to AA induction) with 100μg of Hu3, Hu12, Mt3 or PBS in 100 μl of IFA, or left unvaccinated (AA);and AA was induced on day 0 and arthritis scores were assessed. B. Legswelling measured on day 26 after AA induction.

FIG. 4: Prevention of AA by transfer of Con A-activated splenocytes fromHu3-vaccinated rats. A. Time course of AA. Rats were vaccinated once (onday-7 relative to AA induction) with 100 μg of Hu3 or Mt3 in 100 μl ofIFA and AA was induced on day 0. Splenocytes were collected on day 26after induction of AA, activated for 48 hr with Con A and transferred ivto naïve rats. Three days later, AA was induced in the recipients andarthritis scores were assessed. B. Leg swelling measured on day 26 afterAA induction.

FIG. 5: T-cell responses after DNA vaccination. Lewis rats werevaccinated with pI, pII or pcDNA3 and AA was induced. Twenty-six dayslater, LNC were collected, and the proliferative responses to (A) PPD,(B) HSP65, Mt176-190, Mt3, HSP60 and Hu3 were studied.

FIG. 6: Cytokine secretion after DNA vaccination. Lewis rats werevaccinated with pI, pII or pcDNA3 and AA was induced. Twenty-six dayslater, LNC were collected, stimulated in vitro with PPD, (B) HSP65,Mt176-190, Mt3, HSP60 and Hu3 and the supernatants were tested after 72hr for the amounts of secreted (A) INFγ, (B) IL-10 or (C) TGFβ1.

FIG. 7: T-cell responses after DNA vaccination. Lewis rats werevaccinated with Hu3, Mt3, Hu12 or PBS and AA was induced. Twenty-sixdays later, LNC were collected, and the proliferative responses to (A)PPD, (B) HSP65, Mt176-190, Mt3, HSP60 and Hu3 were studied.

FIG. 8: Cytokine secretion after DNA vaccination. Lewis rats werevaccinated with Hu3, Mt3, Hu12 or PBS, and AA was induced. Twenty-sixdays later, LNC were collected, stimulated in vitro with PPD, (B) HSP65,Mt176-190, Mt3, HSP60 and Hu3 and the supernatants were tested after 72hr for the amounts of secreted (A) INFγ, (13) IL-10 or (C) TGFβ1.

FIG. 9: Modulation of AA by preimmunization with pHSP60 or pHSP65. A.Time course of AA. Rats were vaccinated in the quadriceps three times(on days-40, -26-12 relative to AA induction) with 150 μg of pcDNA3,pMBP, pHSP60 or pHSP65, or left untreated as controls (AA). On day 0 AAwas induced by injecting 1 mg of Mycobacterium tuberculosis (Mt)suspended in 100 μl of IFA, and arthritis scores were assessed every twoor three days starting at day 8 after Mt injection. B. Leg swellingmeasured at day 26 after AA induction.

FIG. 10: Proliferative responses to HSP60, HSP65 and PPD in pHSP60-,pMBP and pcDNA3-vaccinated animals. Female Lewis rats were vaccinated inthe quadriceps three times (on days-40, -26-12 relative to AA induction)with 150 μg of pHSP60, pMBP or pcDNA3, beginning 5 days after i.m.injection of 200 μl of cardiotoxin 10 μM. A group was left untreated asa control. Animals were killed and LNC were collected on day-1 prior tothe induction of AA.

FIG. 11: T-cell responses in AA rats vaccinated with pcDNA3. Animalsvaccinated with pcDNA3 were killed on day 26 after induction of AA andtheir LNC were collected and stimulated in vitro for 72 hrs in thepresence of different concentrations of antigen. The release of IFNγ andIL-10 was studied.

FIG. 12: Effect of vaccination on cytokine secretion. Animals vaccinatedwith pHSP60, pHSP65 or Hu3 were killed on day 26 after induction of AAand their LNC were collected. The cells were stimulated for 72 hrs inthe presence of different antigens, and the proliferation (FIG. 12A orthe release of IFNγ (FIG. 12B, IL-10 (FIG. 12C and TGFβ1 (FIG. 12D werestudied and are illustrated

FIG. 13: Inhibition of AA by preimmunization with pHSP70 or pHSP90. A.Time course of AA. B. Maximal arthritis score. C. Day of onset. D.Difference in leg swelling measured at day 26 after AA induction.

FIG. 14. Humoral response in DNA vaccinated rats. A. Antibodies to HSP70in pHSP70-immunized rats. B. Antibodies to HSP90 in pHSP90-immunizedrats. The day of induction of AA was considered day 0.

FIG. 15. T-cell response to the immunizing antigen in DNA-vaccinatedrats. A. Proliferative response to HSP70 in pHSP70-immunized rats. Theresults are presented as the mean±SD of the stimulation index (SI) inquadruplicate cultures. B. Proliferative response to HSP90 inpHSP90-immunized rats. The results are presented as the mean±SD of theSI in quadruplicate cultures.

FIG. 16. Cytokine secretion in response to stimulation with theimmunizing antigen in DNA-vaccinated rats. Draining lymph node cellswere stimulated for 72 hrs in the presence of HSP70 or HSP90, and thecontent of IFNγ(A) or IL-10 (B) was determined in the supernatants bycapture ELISA.

FIG. 17: Effect of DNA vaccination on T-cell proliferation following AAinduction. The results are expressed as the percent change inproliferation relative to the responses of untreated rats, 26 after theinduction of AA.

FIG. 18: Effect of DNA vaccination on cytokine secretion following AAinduction. DLN cells were stimulated for 72 hrs in the presence of HSP70or HSP90, and the content of IFNγ (A), IL-10 (B) or TGFβ1 (C) wasdetermined in the supernatants by capture ELISA.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention it is now disclosed that it ispossible to treat or prevent T cell-mediated inflammatory autoimmunediseases by using DNA vaccines encoding a heat shock protein, or activefragments thereof. The compositions and methods of the present inventionare effective in any T-cell mediated inflammatory autoimmune diseaseincluding but not limited to: rheumatoid arthritis, collagen IIarthritis, multiple sclerosis, autoimmune neuritis, systemic lupuserythematosus, psoriasis, juvenile onset diabetes, Sjogren's disease,thyroid disease, sarcoidosis, autoimmune uveitis, inflammatory boweldisease (Crohn's and ulcerative colitis) or autoimmune hepatitis.

The present invention is based in part on studies of the role of DNAvaccines encoding a heat shock protein, or fragments thereof in adjuvantinduced arthritis in experimental rats. Specifically, the presentinvention is based on the unexpected discovery that certain DNAconstructs encoding specific heat shock proteins, such as HSP60, HSP70or HSP90, or active fragments thereof are useful in decreasing thesymptoms associated with arthritis. The protective effect of these DNAconstructs was reflected for example by a significant reduction in ankleswelling.

It is now disclosed that it is possible to treat or prevent Tcell-mediated diseases by using DNA vaccines encoding mammalian heatshock proteins or active fragments thereof. The present invention isbased in part on studies of the role of the immune response to HSP60 inadjuvant induced arthritis in experimental rats, using DNA vaccinesencoding human HSP60, human HSP70, human HSP90 or active fragmentsthereof The results led to the identification of novel constructsencoding at least part of the HSP60 sequence that could effectivelysuppress AA. In addition, specific HSP60 fragments were found to beeffective in suppressing AA. The immune effects associated with specificDNA or peptide suppression of AA were complex and included enhancedT-cell proliferation to a variety of disease-associated antigens.Effective vaccination with HSP60 DNA fragments or the HSP60 peptide ledto up-regulation of IFNγ secretion to HSP60 and, concomitantly todown-regulation of IFNγ secretion to mycobacterial HSP65 epitopes. Therewere also variable changes in the profiles of IL-10 secretion to thoseantigens. The production of TGFβ1, however, was enhanced to both HSP60and HSP65 epitopes. The regulation of AA might be due to the inductionof regulatory T-cells directed to HSP60, secreting both Th1 and Th2cytokines that shifted the immune response towards mycobacterialantigens to a Th2 non-pathogenic response.

The present invention provides an effective method of DNA vaccinationfor T cell-mediated inflammatory autoimmune diseases, which avoids manyof the problems associated with the previously suggested methods oftreatment. By vaccinating, rather than passively administeringheterologous antibodies, the host's own immune system is mobilized tosuppress the autoaggressive T cells. Thus, the suppression is persistentand may involve any and all immunological mechanisms in effecting thatsuppression. This multi-faceted response is more effective than theuni-dimensional suppression achieved by passive administration ofmonoclonal antibodies or extant-derived regulatory T cell clones.

In one aspect, the present invention is related to novel recombinantconstructs comprising a nucleic acid sequence corresponding to mammalianheat shock proteins, the nucleic acid sequence being operatively linkedto at least one transcription control element. Preferably, therecombinant constructs of the present invention correspond to human heatshock proteins. However, recombinant constructs corresponding to the rator mouse heat shock proteins may also be used in the present invention.

The nucleic acid sequence corresponding to mammalian heat shock proteinsmay include DNA, RNA, or derivatives of either DNA or RNA. An isolatednucleic acid sequence encoding heat shock proteins can be obtained fromits natural source, either as an entire (i.e., complete) gene or aportion thereof A nucleic acid molecule can also be produced usingrecombinant DNA technology (e.g., polymerase chain reaction (PCR)amplification, cloning) or chemical synthesis. Nucleic acid sequencesinclude natural nucleic acid sequences and homologues thereof,including, but not limited to, natural allelic variants and modifiednucleic acid sequences in which nucleotides have been inserted, deleted,substituted, and/or inverted in such a manner that such modifications donot substantially interfere with the nucleic acid molecule's ability toencode a functional heat shock protein or an active fragment thereof.

A nucleic acid sequence homologue can be produced using a number ofmethods known to those skilled in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LabsPress, 1989). For example, nucleic acid sequences can be modified usinga variety of techniques including, but not limited to, classicmutagenesis techniques and recombinant DNA techniques, such assite-directed mutagenesis, chemical treatment of a nucleic acid moleculeto induce mutations, restriction enzyme cleavage of a nucleic acidfragment, ligation of nucleic acid fragments, polymerase chain reaction(PCR) amplification and/or mutagenesis of selected regions of a nucleicacid sequence, synthesis of oligonucleotide mixtures and ligation ofmixture groups to “build” a mixture of nucleic acid molecules andcombinations thereof. Nucleic acid molecule homologues can be selectedfrom a mixture of modified nucleic acids by screening for the functionof the protein encoded by the nucleic acid.

The present invention includes a nucleic acid sequence operativelylinked to one or more transcription control sequences to form arecombinant molecule. The phrase “operatively linked” refers to linkinga nucleic acid sequence to a transcription control sequence in a mannersuch that the molecule is able to be expressed when transfected (i.e.,transformed, transduced or transfected) into a host cell. Transcriptioncontrol sequences are sequences which control the initiation,elongation, and termination of transcription. Particularly importanttranscription control sequences are those which control transcriptioninitiation, such as promoter, enhancer, operator and repressorsequences. Suitable transcription control sequences include anytranscription control sequence that can function in at least one of therecombinant cells of the present invention. A variety of suchtranscription control sequences are known to those skilled in the art.Preferred transcription control sequences include those which functionin animal, bacteria, helminth, insect cells, and preferably in animalcells. More preferred transcription control sequences include, but arenot limited to RSV control sequences, CMV control sequences, retroviralLTR sequences, SV-40 control sequences and β-actin control sequences aswell as other sequences capable of controlling gene expression ineukaryotic cells. Additional suitable transcription control sequencesinclude tissue-specific promoters and enhancers (e.g., T cell-specificenhancers and promoters). Transcription control sequences of the presentinvention can also include naturally occurring transcription controlsequences naturally associated with a gene encoding a heat shock proteinof the present invention.

The present invention is further related to an expression vectorcomprising the recombinant constructs of the present invention. Suitableeukaryotic expression vector is for example: pcDNA3, pcDNA3.1(+/−),pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1,pCI, pBK-RSV, pBK-CMV, pTRES or their derivatives.

According to the present invention, a host cell can be transfected invivo (i.e., in an animal) or ex vivo (i.e., outside of an animal).Transfection of a nucleic acid molecule into a host cell can beaccomplished by any method by which a nucleic acid molecule can beinserted into the cell. Transfection techniques include, but are notlimited to, transfection, electroporation, microinjection, lipofection,adsorption, and protoplast fusion. Preferred methods to transfect hostcells in vivo include lipofection and adsorption.

It may be appreciated by one skilled in the art that use of recombinantDNA technologies can improve expression of transfected nucleic acidmolecules by manipulating, for example, the number of copies of thenucleic acid molecules within a host cell, the efficiency with whichthose nucleic acid molecules are transcribed, the efficiency with whichthe resultant transcripts are translated, and the efficiency ofpost-translational modifications. Recombinant techniques useful forincreasing the expression of nucleic acid molecules of the presentinvention include, but are not limited to, operatively linking nucleicacid molecules to high-copy number plasmids, integration of the nucleicacid molecules into one or more host cell chromosomes, addition ofvector stability sequences to plasmids, substitutions or modificationsof transcription control signals (e.g., promoters, operators,enhancers), substitutions or modifications of translational controlsignals (e.g., ribosome binding sites, Shine-Dalgarno sequences),modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, and deletion ofsequences that destabilize transcripts.

According to yet another aspect of the present invention there isprovided a pharmaceutical composition suitable for effecting the abovemethods of the present invention. The composition includes a recombinantconstruct including an isolated nucleic acid sequence encoding a heatshock protein or a fragment thereof, the nucleic acid sequence beingoperatively linked to one or more transcription control sequences, and apharmaceutically acceptable carrier.

In one embodiment of the invention, the composition is useful fortreating a T cell-mediated inflammatory autoimmune disease such asmultiple sclerosis, rheumatoid arthritis, collagen II arthritis,autoimmune neuritis, systemic lupus erythematosus, psoriasis, juvenileonset diabetes, Sjogren's disease, thyroid disease, sarcoidosis,autoimmune uveitis, inflammatory bowel disease (Crohn's and ulcerativecolitis) or autoimmune hepatitis.

The therapeutic composition of the invention is administered to anindividual in need of said treatment. According to still furtherfeatures in the described preferred embodiments the individual isselected from the group consisting of humans, dogs, cats, sheep, cattle,horses and pigs.

In another embodiment of the present invention, a therapeuticcomposition further comprises a pharmaceutically acceptable carrier. Asused herein, a “carrier” refers to any substance suitable as a vehiclefor delivering a nucleic acid sequence of the present invention to asuitable in vivo site. As such, carriers can act as a pharmaceuticallyacceptable excipient of a therapeutic composition containing a nucleicacid molecule of the present invention. Preferred carriers are capableof maintaining a nucleic acid molecule of the present invention in aform that, upon arrival of the nucleic acid molecule to a cell, thenucleic acid molecule is capable of entering the cell and beingexpressed by the cell. Carriers of the present invention include: (1)excipients or formularies that transport, but do not specifically targeta nucleic acid molecule to a cell (referred to herein as non-targetingcarriers); and (2) excipients or formularies that deliver a nucleic acidmolecule to a specific site in an animal or a specific cell (i.e.,targeting carriers). Examples of non-targeting carriers include, but arenot limited to water, phosphate buffered saline, Ringer's solution,dextrose solution, serun-containing solutions, Hank's solution, otheraqueous physiologically balanced solutions, oils, esters and glycols.Aqueous carriers can contain suitable auxiliary substances required toapproximate the physiological conditions of the recipient, for example,by enhancing chemical stability and isotonicity.

Suitable auxiliary substances include, for example, sodium acetate,sodium chloride, sodium lactate, potassium chloride, calcium chloride,and other substances used to produce phosphate buffer, Tris buffer, andbicarbonate buffer. Auxiliary substances can also include preservatives,such as thimerosal, m- and o-cresol, formalin and benzol alcohol.Preferred auxiliary substances for aerosol delivery include surfactantsubstances non-toxic to an animal, for example, esters or partial estersof fatty acids containing from about six to about twenty-two carbonatoms. Examples of esters include, caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric, and oleic acids. Other carrierscan include metal particles (e.g., gold particles) for use with, forexample, a biolistic gun through the skin. Therapeutic compositions ofthe present invention can be sterilized by conventional methods.

Targeting carriers are herein referred to as “delivery vehicles”.Delivery vehicles of the present invention are capable of delivering atherapeutic composition of the present invention to a target site in ananimal. A “target site” refers to a site in an animal to which onedesires to deliver a therapeutic composition. For example, a target sitecan be a cancer cell, a tumor, or a lesion caused by an infectiousagent, or an area around such cell, tumor or lesion, which is targetedby direct injection or delivery using liposomes or other deliveryvehicles. Examples of delivery vehicles include, but are not limited to,artificial and natural lipid-containing delivery vehicles. Naturallipid-containing delivery vehicles include cells and cellular membranes.Artificial lipid-containing delivery vehicles include liposomes andmicelles. A delivery vehicle of the present invention can be modified totarget to a particular site in an animal, thereby targeting and makinguse of a nucleic acid molecule of the present invention at that site.Suitable modifications include manipulating the chemical formula of thelipid portion of the delivery vehicle and/or introducing into thevehicle a compound capable of specifically targeting a delivery vehicleto a preferred site, for example, a preferred cell type. Specificallytargeting refers to causing a delivery vehicle to bind to a particularcell by the interaction of the compound in the vehicle to a molecule onthe surface of the cell. Suitable targeting compounds include ligandscapable of selectively (i.e., specifically) binding another molecule ata particular site. Examples of such ligands include antibodies,antigens, receptors and receptor ligands. For example, an antibodyspecific for an antigen found on the surface of a cancer cell can beintroduced to the outer surface of a liposome delivery vehicle so as totarget the delivery vehicle to the cancer cell. Tumor cell ligandsinclude ligands capable of binding to a molecule on the surface of atumor cell. Manipulating the chemical formula of the lipid portion ofthe delivery vehicle can modulate the extracellular or intracellulartargeting of the delivery vehicle. For example, a chemical can be addedto the lipid formula of a liposome that alters the charge of the lipidbilayer of the liposome so that the liposome fuses with particular cellshaving particular charge characteristics.

According to one embodiment, fat emulsions may be used as a vehicle forDNA vaccines. Two examples of such emulsions are the availablecommercial fat emulsions known as Intralipid and Lipofundin.“Intralipid” is a registered trademark of Kabi Pharmacia, Sweden, for afat emulsion for intravenous nutrition, described in U.S. Pat. No.3,169,094. “Lipofundin” is a registered trademark of B. Braun Melsungen,Germany. Both contain soybean oil as fat (100 or 200 g in 1,000 mldistilled water: 10% or 20%, respectively). Egg-yolk phospholipids areused as emulsifiers in Intralipid (12 g/l distilled water) and egg-yolklecithin in Lipofundin (12 g/l distilled water). Isotonicity resultsfrom the addition of glycerol (25 g/l) both in Intralipid andLipofundin.

According to another embodiment, the delivery vehicle of the presentinvention may be a liposome. A liposome is capable of remaining stablein an animal for a sufficient amount of time to deliver a nucleic acidsequence of the present invention to a preferred site in the animal. Aliposome of the present invention is preferably stable in the animalinto which it has been administered for at least about 30 minutes, morepreferably for at least about 1 hour and even more preferably for atleast about 24 hours.

A liposome of the present invention comprises a lipid composition thatis capable of fusing with the plasma membrane of the targeted cell todeliver a nucleic acid molecule into a cell. Preferably, thetransfection efficiency of a liposome of the present invention is about0.5 microgram (μg) of DNA per 16 nanomole (nmol) of liposome deliveredto about 10⁶ cells, more preferably about 1.0 μg of DNA per 16 nmol ofliposome delivered to about 10⁶ cells, and even more preferably about2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells.

A preferred liposome of the present invention is between about 100 and500 nanometers (nm), more preferably between about 150 and 450 nm andeven more preferably between about 200 and 400 nm in diameter.

Suitable liposomes for use with the present invention include anyliposome. Preferred liposomes of the present invention include thoseliposomes standardly used in, for example, gene delivery methods knownto those of skill in the art. More preferred liposomes compriseliposomes having a polycationic lipid composition and/or liposomeshaving a cholesterol backbone conjugated to polyethylene glycol.

Complexing a liposome with a nucleic acid sequence of the presentinvention can be achieved using methods standard in the art. A suitableconcentration of a nucleic acid molecule of the present invention to addto a liposome includes a concentration effective for delivering asufficient amount of nucleic acid molecule to a cell such that the cellcan produce sufficient heat shock protein to regulate effector cellimmunity in a desired manner. Preferably, from about 0.1 μg to about 10μg of nucleic acid sequence of the present invention is combined withabout 8 nmol liposomes, more preferably from about 0.5 μg to about 5 μgof nucleic acid molecule is combined with about 8 nmol liposomes, andeven more preferably about 1.0 μg of nucleic acid molecule is combinedwith about 8 nmol liposomes.

According to another embodiment, the delivery vehicle comprises arecombinant cell vaccine. Preferred recombinant cell vaccines of thepresent invention include cell vaccines, in which allogeneic (i.e.,cells derived from a source other than a patient, but that arehistiotype compatible with the patient) or autologous (i.e., cellsisolated from a patient) cells are transfected with recombinantmolecules contained in a therapeutic composition, irradiated andadministered to a patient by, for example, intradermal, intravenous orsubcutaneous injection. Therapeutic compositions to be administered bycell vaccine, include recombinant molecules of the present inventionwithout carrier.

In order to treat an animal with disease, a therapeutic composition ofthe present invention is administered to the animal in an effectivemanner such that the composition is capable of treating that animal fromdisease. For example, a recombinant molecule, when administered to ananimal in an effective manner, is able to stimulate effector cellimmunity in a manner that is sufficient to alleviate the diseaseafflicting the animal. According to the present invention, treatment ofa disease refers to alleviating a disease and/or preventing thedevelopment of a secondary disease resulting from the occurrence of aprimary disease. An effective administration protocol (i.e.,administering a therapeutic composition in an effective manner)comprises suitable dose parameters and modes of administration thatresult in treatment of a disease. Effective dose parameters and modes ofadministration can be determined using methods standard in the art for aparticular disease. Such methods include, for example, determination ofsurvival rates, side effects (i.e., toxicity) and progression orregression of disease. In particular, the effectiveness of doseparameters and modes of administration of a therapeutic composition ofthe present invention when treating cancer can be determined byassessing response rates. Such response rates refer to the percentage oftreated patients in a population of patients that respond with eitherpartial or complete remission.

In accordance with the present invention, a suitable single dose size isa dose that is capable of treating an animal with disease whenadministered one or more times over a suitable time period. Doses canvary depending upon the disease being treated. Doses of a therapeuticcomposition of the present invention suitable for use with directinjection techniques can be used by one of skill in the art to determineappropriate single dose sizes for systemic administration based on thesize of an animal. A suitable single dose of a therapeutic compositionis a sufficient amount of heat shock protein-encoding recombinantsequence to reduce, and preferably eliminate, the T-cell mediatedautoimmune disease following transfection of the recombinant moleculesinto cells. A preferred single dose of heat shock protein-encodingrecombinant molecule is an amount that, when transfected into a targetcell population leads to the production of from about 250 femtograms(fg) to about 1 μg, preferably from about 500 fg to about 500 picogram(pg), and more preferably from about 1 pg to about 100 pg of a heatshock protein or fragment thereof per transfected cell.

A preferred single dose of heat shock protein-encoding recombinantmolecule complexed with liposomes, is from about 100 μg of total DNA per800 nmol of liposome to about 2 mg of total recombinant molecules per 16micromole (μmol) of liposome, more preferably from about 150 μg per 1.2μmol of liposome to about 1 mg of total recombinant molecules per 8 μmolof liposome, and even more preferably from about 200 μg per 2 μmol ofliposome to about 400 μg of total recombinant molecules per 3.2 μmol ofliposome.

A preferred single dose of heat shock protein-encoding recombinantmolecule in a non-targeting carrier to administer to an animal, is fromabout 100 μg to about 4 mg of total recombinant molecules, morepreferably from about 150 μg to about 3 mg of total recombinantmolecules, and even more preferably from about 200 μg to about 2 mg oftotal recombinant molecules.

It will be obvious to one of skill in the art that the number of dosesadministered to an animal is dependent upon the extent of the diseaseand the response of an individual patient to the treatment. Thus, it iswithin the scope of the present invention that a suitable number ofdoses includes any number required to cause regression of a disease. Apreferred protocol is monthly administrations of single doses (asdescribed above) for up to about 1 year. A preferred number of doses ofa therapeutic composition comprising heat shock protein-encodingrecombinant molecule in a non-targeting carrier or complexed withliposomes is from about 1 to about 10 administrations per patient,preferably from about 2 to about 8 administrations per patient, and evenmore preferably from about 3 to about 5 administrations per person.Preferably, such administrations are given once every 2 weeks untilsigns of remission appear, then once a month until the disease is gone.

A therapeutic composition is administered to an animal in a fashion toenable expression of the administered recombinant molecule of thepresent invention into a curative protein in the animal to be treatedfor disease. A therapeutic composition can be administered to an animalin a variety of methods including, but not limited to, localadministration of the composition into a site in an animal, and systemicadministration.

Therapeutic compositions to be delivered by local administrationinclude: (a) recombinant molecules of the present invention in anon-targeting carrier (e.g., as “naked” DNA molecules, such as istaught, for example in Wolff et al., 1990, Science 247, 1465-1468); and(b) recombinant molecules of the present invention complexed to adelivery vehicle of the present invention. Suitable delivery vehiclesfor local administration comprise liposomes or emulsions. Deliveryvehicles for local administration can further comprise ligands fortargeting the vehicle to a particular site.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site. Systemicadministration is particularly advantageous when organs, in particulardifficult to reach organs (e.g., heart, spleen, lung or liver) are thetargeted sites of treatment.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference in itsentirety). Oral delivery can be performed by complexing a therapeuticcomposition of the present invention to a carrier capable ofwithstanding degradation by digestive enzymes in the gut of an animal.Examples of such carriers, include plastic capsules or tablets, such asthose known in the art. Topical delivery can be performed by mixing atherapeutic composition of the present invention with a lipophilicreagent (e.g., DMSO) that is capable of passing into the skin.

Suitable embodiments, single dose sizes, number of doses and modes ofadministration of a therapeutic composition of the present inventionuseful in a treatment method of the present invention are disclosed indetail herein.

A therapeutic composition of the present invention is also advantageousfor the treatment of autoimmune diseases in that the compositionsuppresses the harmful stimulation of T cells by autoantigens (i.e., a“self”, rather than a foreign antigen). Heat shock protein-encodingrecombinant molecules in a therapeutic composition, upon transfectioninto a cell, produce a heat shock protein or a fragment thereof thatreduces the harmful activity of T cells involved in an autoimmunedisease. A preferred therapeutic composition for use in the treatment ofautoimmune disease comprises heat shock protein-encoding recombinantmolecule of the present invention. A more preferred therapeuticcomposition for use in the treatment of autoimmune disease comprisesheat shock protein-encoding recombinant molecule combined with anon-targeting carrier of the present invention, preferably saline orphosphate buffered saline.

A single dose of heat shock protein-encoding nucleic acid molecule in anon-targeting carrier to administer to an animal to treat an autoimmunedisease is from about 0.1 μg to about 200 μg of total recombinantmolecules per kilogram (kg) of body weight, more preferably from about0.5 μg to about 150 μg of total recombinant molecules per kg of bodyweight, and even more preferably from about 1 μg to about 10 μg of totalrecombinant molecules per kg of body weight.

The number of doses of heat shock protein-encoding recombinant moleculein a non-targeting carrier to be administered to an animal to treat anautoimmune disease is an injection about once every 6 months, morepreferably about once every 3 months, and even more preferably aboutonce a month.

A preferred method to administer a therapeutic composition of thepresent invention to treat an autoimmune disease is by direct injection.Direct injection techniques are particularly important in the treatmentof an autoimmune disease. Preferably, a therapeutic composition isinjected directly into muscle cells in a patient, which results inprolonged expression (e.g., weeks to months) of a recombinant moleculeof the present invention. Preferably, a recombinant molecule of thepresent invention in the form of “naked DNA” is administered by directinjection into muscle cells in a patient.

It is to be noted that the compositions and methods of the presentinvention do not include the obligatory presence of the CpG motifdisclosed in WO 02/16549, in DNA vaccines suitable for the treatment ofongoing autoimmune diseases.

The following examples are presented in order to more fully illustratecertain embodiments of the invention. They should in no way, however, beconstrued as limiting the broad scope of the invention. One skilled inthe art can readily devise many variations and modifications of theprinciples disclosed herein without departing from the scope of theinvention.

EXAMPLES

Materials and Methods

Animals

Female Lewis rats were raised and maintained under pathogen-freeconditions in the Animal Breeding Center of The Weizmann Institute ofScience. One- to two-month old rats were used for DNA vaccination andpeptide-vaccination experiments. The experiments were performed underthe supervision and guidelines of the Animal Welfare Committee.

Antigens and Adjuvants

Peptides were synthesized as previously described (15). The HSP60peptides used in these studies are listed in Table I. Two HSP65 peptideswere also used: Mt176-190, EESNTFGLQLELTEG (16) and Mt3,AYDEEARRGLERGLNALADA. Purified recombinant HSP65 was generously providedby Prof. Ruurd van der Zee (Institute of Infectious Diseases andImmunology, Faculty of Veterinary Medicine, Utrecht, The Netherlands).Recombinant HSP60 was prepared in our laboratory as described (11). M.tuberculosis Strain H37Ra and incomplete Freund's adjuvant (IFA) werepurchased from Difco (Detroit, Mich., USA). Tuberculin purified proteinderivative (PPD) was provided by the Statens Serumistitut (Copenhagen,Denmark). Ovalbumin (OVA) and Concanavalin A (Con A) were purchased fromSigma (Rehovot, Israel).

DNA Plasmids

The vector containing the human hsp60 gene (pHSP60) has been described(17). The construct encoding Mycobacterium leprae HSP65 (pHSP65) waskindly provided by Dr. Douglas Lowrie (Medical Research Council, London,UK). Five fragments of the human hsp60 gene were amplified by PCR fromhsp60 cDNA in pGEM (Promega, Madison, Wis., USA) using specificoligonucleotides containing restriction sites for the enzymes BamHI(oligonucleotide 5′) or HindIII (oligonucleotide 3′), and cloned intothe pcDNA3 vector (Invitrogen, NV, Leek, The Netherlands) using standardmolecular biology techniques (Table II). The 5′ oligonucleotide alsoincluded an ATG sequence necessary for protein translation. The plasmidswere sequenced to confirm correct insertion of the cDNA and transcribedin vitro to check that they are functional (data not shown).

Plasmid DNA was prepared in large scale and injected after pretreatmentwith cardiotoxin (Sigma, Rehovot, Israel). Briefly, rats were vaccinatedin the quadriceps three times (on days-40, -26-12 relative to AAinduction) with 150 μg of pcDNA3, pI, pI, pIII, pIV or pV. Endotoxinlevels were checked by Limulus Amoebocyte Lysate and found always to beunder acceptable levels for in vivo use (less than 0.02 EU/μg DNA). AAwas induced 12 days after the last injection of DNA. The empty vectorpcDNA3 was used as a DNA vaccination control.

AA Induction and Assessment

AA was induced using 1 mg per rat of heat-killed Mt strain H37Ra(Difco). The day of AA induction was designated as day 0, and diseaseseverity was assessed by direct observation of all 4 limbs in eachanimal. A relative score between 0 and 4 was assigned to each limb,based on the degree of joint inflammation, redness and deformity; thusthe maximum possible score for an individual animal was 16. Arthritiswas also quantified by measuring hind limb diameter with a caliper.Measurements were taken on the day of AA induction and 26 days later,and they are presented as the mean±SEM of the difference between the twovalues. The person who scored the disease was blinded to the identity ofthe groups.

T-Cell Proliferation

Popliteal and inguinal lymph node cells (LNC) taken 26 days after theinduction of AA were cultured in quadruplicates in 200 μl round bottommicrotiter wells (Costar Corp., Cambridge, USA) at 2×10⁵ cells per wellwith or without antigen. The T-cell mitogen Concanavalin A (Con A) wasused as a positive control for T-cell proliferation. Cultures wereincubated for 96 hrs at 37° C. in a humidified atmosphere of 5% CO₂.T-cell responses were detected by the incorporation of[methyl-³H]-thymidine (Amersham, Buckinghamshire, UK; 1 μCi/well), whichwas added to the wells for the last 18 hours. The stimulation index (SI)was computed as the ratio of the mean c.p.m. of antigen- ormitogen-containing wells to control wells cultured with medium alone.

Transfer of Cells

Spleen cells were prepared from peptide-vaccinated rats 26 days afterthe induction of AA. The splenocytes (10⁷ cells per ml) were activatedwith 2.5 μg/ml of Con A for 48 hr at 37° C. in a humidified atmosphereof 5% CO₂. The cells were washed with sterile PBS and injected iv intonaïve rats (5×10⁷ cells per rat). Three days after the transfer of thesplenocytes, AA was induced.

Cytokine Assays

Supernatants were collected after 72 hrs of stimulation with each of theantigens tested. Rat IL-10 and IFNγ were quantitated in culturesupernatants by enzyme-linked immunosorbent assay (ELISA) usingPharmingen's OPTEIA kit (Pharmingen, San Diego, USA). Rat TGFβ1 wasquantified using the TGFβ1 E_(max)® ImmunoAssay System (Promega,Madison, USA) according to the manufacturer's instructions. Cytokinelevels in supernatants are expressed as pg/ml based on calibrationcurves constructed using recombinant cytokines as standards. The lowerlimits of detection for the experiments described in this paper were 15pg/mL for TGFβ1, IL-10 and IFNγ.

Statistical Significance

The InStat 2.01 program was used for statistical analysis. Student'st-test and the Mann-Whitney test were carried out to assay significantdifferences between the different experimental groups.

Example 1 HSP60 DNA Fragments Inhibit AA

To learn whether fragments of HSP60 DNA could inhibit AA, the cDNAcorresponding to the human hsp60 gene was divided into five fragments,each with a 30 pb overlap, and each was cloned into the pcDNA3 vector(Table II). In this way, five constructs corresponding to HSP60-derivedfragments were generated overlapping by 10 aa: pI, aa 1-140; pII, aa130-260; pIII, aa 250-410, pIV, aa 400-470 and pV, aa 460-540 (Table II.Lewis rats were vaccinated with one of the HSP60 fragment constructs orwith pcDNA3 as a control, and AA was induced. FIG. 1A shows that therats vaccinated with pI or pII manifested significantly decreasedarthritis compared to rats vaccinated with constructs pIII, pIV or pV orwith control pcDNA3. The protective effect of the vaccination withconstructs pI and pII was also reflected by a significant reduction inankle swelling (FIG. 1B), and by a reduction of the mean maximal score,which was lower in those rats vaccinated with pI and pII (p=0.0007 andp=0.0003, respectively, compared to rats vaccinated with pcDNA3).

Example 2 In Vitro Proliferation of LNC Isolated from pHSP60- orPI-Vaccinated Rats in Response to Various HSP60 Peptides

To learn whether the suppression of AA by DNA vaccination with pHSP60,pI or pII was associated with T-cell reactivity to a specific HSP60epitope, the proliferation (Stimulation Index, SI) of LNC isolated frompHSP60-vaccinated rats was studied in response to a panel of overlappingpeptides spanning the region of human HSP60 encoded by pI and pII (aa1-275; Table I). Controls were LNC prepared from rats vaccinated withpcDNA3 or pHSP65 and challenged with Mt suspended in IFA to induce AA.FIG. 2A shows that only peptide Hu3 (aa 31-50 of human HSP60) induced asignificant response in LNC taken from pHSP60-vaccinated rats; cellsprepared from pHSP65 or pcDNA-vaccinated rats did not respond to Hu3.Note that the sequence of the HSP60 protein in the region 31-50 isidentical in rat and human HSP60; thus Hu3 is a self-peptide (TableIII). FIG. 2B shows a dose-dependant proliferative response to Hu3 usingLNC isolated from pHSP60-vaccinated rats. No significant responses tothe control peptide Hu12 (aa 166-185 of human HSP60) were detected. Toconfirm these results, T-cell proliferative responses were studied inLNC taken 26 days after the induction of AA from rats vaccinated withpI, pII or pcDNA3. FIG. 2C shows that Hu3, but not its mycobacterialhomologue Mt3, triggered a significant proliferation of LNC taken frompI-immunized rats, but not by LNC from rats vaccinated with pII orpcDNA3. Non of the HSP60 peptides was specifically recognized by LNCtaken from pI-immunized rats. In summary, these results show thatpHSP60- and pI-vaccinated rats manifested up-regulated T-cell responsesto the Hu3 peptide of HSP60.

Example 3 Peptide Hu3 Inhibits AA

To establish a link between the immune response to Hu3 and prevention ofAA, rats were vaccinated with Hu3, or with its mycobacterial counterpartMt3 (Table III) or with Hu12 (Table I) as controls. Each rat received asingle i.p. dose of 100 μg of peptide in IFA seven days (day-7) beforethe induction of AA (day 0). FIG. 3A shows that the Hu3-vaccinated ratsdeveloped significantly decreased disease compared to non-immunized ratsor rats vaccinated with PBS, Hu12 or Mt3. This reduction in AA was alsoreflected by a significant reduction in ankle swelling (14.2±4.7 inHu3-vaccinated rats vs 32.2±3.5 in PBS/IFA-vaccinated rats, p=0.02; FIG.3B). Therefore, vaccination with Hu3 prevents AA.

Example 4 Adoptive Transfer of Peptide-Induced Regulation

To learn whether the protection triggered by Hu3-vaccination could beadoptively transferred by activated T-cells, splenocytes prepared fromHu3 vaccinated rats were stimulated in vitro for 2 days with the T-cellmitogen Con A, washed, and injected iv (5×10⁷ cells per rat) into naïverats. Only the recipients of cells taken from Hu3-vaccinated rats wereprotected against the subsequent induction of AA (FIGS. 4A and 4B). Noprotection was seen in rats that had received Con A activated cells fromMt3 injected rats. Thus, inhibition of AA by vaccination with Hu3 couldbe adoptively transferred by activated T-cells.

Example 5 In Vitro Proliferation of LNC Isolated from PI- orPII-Vaccinated Rats in Response to Various Mycobacterial Antigens, HSP60or its Hu3 Peptide

To study the mechanism associated with the inhibition of AA by DNAvaccination with pI or pII, the T-cell responses of immunized rats wasanalysed 26 days after the induction of AA. The LNC were stimulated invitro with a collective of mycobacterial antigens known to be associatedwith AA: HSP65, PPD, Mt176-90 (which contains the 180-188 epitope ofHSP65). The immune response directed to mammalian HSP60, its regulatorypeptide Hu3 and the HSP60-derived peptide Hu12 as a control was studiedas well. OVA was included as a control antigen. None of the experimentalgroups showed significant responses to OVA or Hu12, and they did notdiffer in their response to Con A (data not shown). Nevertheless,inhibition of AA by DNA vaccination with the pI or pII constructs wasassociated with the up-regulation of the T-cell proliferative responsesdirected against the panel of mycobacterial antigens (PPD, HSP65 andMt176-190) (FIG. 5A). FIG. 5B depicts the proliferative responses toHSP60 and its Hu3 peptide. It can be seen that both pI and pII inducedsignificant T-cell responses to HSP60, however, only LNC frompI-vaccinated rats manifested reactivity to Hu3.

Example 6 Cytokine Secretion by LNC Taken from Rats Vaccinated with pI,pII or pcDNA3

Cytokine secretion by LNC taken from rats vaccinated with pI, pII orpcDNA3 was determined. Inhibition of AA by DNA vaccination with pI wasassociated with a decrease in IFNγ secretion (FIG. 6A), and an increasein IL-10 and TGFβ1 secretion upon stimulation with PPD, HSP65 orMt176-190 (FIGS. 6B and 6C).

LNC from pII-vaccinated rats also showed a decrease in IFNγ secretionupon stimulation with PPD, HSP65 and Mt176-190 (FIG. 6A), however IL-10secretion was only detected after activation with HSP65 while TGFβ1secretion was only detected following activation with Mt176-190 or PPD(FIGS. 6B and 6C). Note that cells from both pI or pII-immunized ratssecreted detectable amounts of TGFβ1 upon activation with Mt3. Thus,protection from AA by DNA vaccination with pI or pII was associated withdecreased IFNγ secretion and a concomitant increase in IL-10 and/orTGFβ1 secretion upon stimulation with the mycobacterial antigens PPD,HSP65 or Mt176-190 (FIGS. 6A, 6B and 6C).

In addition to the responses to mycobacterial antigens, the effects ofDNA vaccination with HSP60 fragments on the responses to HSP60 and Hu3was studied. IFNγ was not secreted in response to HSP60 or Hu3 by theLNC of either the pI or pII-immunized rats (FIG. 6A). LNC taken from pIimmunized rats secreted both IL-10 and TGFβ1 in response to HSP60 or Hu3(FIGS. 6B and 6C). LNC cells taken from pII-vaccinated rats, incontrast, secreted TGFβ1 upon activation with HSP60, but IL-10 was notdetected. Therefore, both pI or pII vaccination induced the secretion ofTGFβ1 in response to HSP60. However only pI triggered the secretion ofIL-10.

Example 7 In Vitro Proliferation of LNC Isolated from Hu3-VaccinatedRats in Response to Various Mycobacterial Antigens or HSP60

The T-cell responses after induction of AA in rats that had beenvaccinated with peptides Hu3, Hu12 or Mt3 was examined. All threepeptides were immunogenic; significant and specific T-cell responsescould be detected in the immunized rats to each immunogen (FIG. 7A).However, only the LNC taken from Hu3-vaccinated rats showed up-regulatedT-cell proliferative responses to the mycobacterial antigens PPD, HSP65and Mt176-190 (FIG. 7B). Furthermore, vaccination with Hu3 led to theinduction of a specific response to HSP60 (FIG. 7B). None of theexperimental groups showed significant responses to OVA, and they didnot differ in their response to Con A (data not shown).

Example 8 Cytokine Secretion by LNC Taken from Rats Vaccinated with Hu3

Vaccination with Hu3 led to a reduction in IFNγ secretion (FIG. 8A), andto a concomitant increase in the secretion of IL-10 (FIG. 8B) and TGFβ1(FIG. 8C) upon stimulation with PPD, HSP65 or Mt176-190. Hu3-vaccinationalso led to the induction of T-cells that secreted IFNγ, IL-10 and TGFβ1in response to Hu3. The response to whole HSP60 was predominantly TGFβ1.

Example 9 Human pHPS60 is More Effective than Mycobacterial pHSP65 inInhibiting AA

The effects on AA of vaccination with DNA encoding human pHSP60 comparedto mycobacterial HSP65 was examined. The construct encoding thefull-length human HSP60 (pHSP60) has more than 97% percent identity atthe amino acid level with its rat counterpart. A construct encoding forthe full-length HSP65 of Mycobacterium was used as well. Two controlconstructs were used: an empty vector (pcDNA3), and a construct encodingmurine Myelin Basic Protein (pMBP). FIG. 9 a shows that vaccination withpcDNA3 or pMBP did not have any effect on AA. In contrast, ratsimmunized with pHSP60 or pHSP65 manifested a significantly milderarthritis. Inhibition of AA was also reflected as a diminished swellingof the ankle, as shown in FIG. 9 b. It can be seen that pHSP60 was moreeffective than pHSP65 in modulating the autoimmune process. Thedifference between pHSP60 and pHSP65 was statistically significant withregard to the maximal AA index (2.25±0.65 vs. 7.67±1.83, p=0.02), andleg swelling (10.64±3.43 vs. 27.5±6.35, p=0.03).

Example 10 In Vitro Proliferation of LNC Isolated from Rats after DNAVaccination with pHSP60

The immune response induced by pHSP60-vaccination alone was studiedbefore disease induction. Spleen cells were prepared 10 days after theadministration of the third dose of the DNA-vaccine, and theproliferative response upon in vitro stimulation with different antigenswas studied. FIG. 10 shows that pHSP60-vaccination induced a significantproliferative response to HSP60, but not to HSP65, MBP or PPD, whilecells from pMBP-treated rats only proliferated in response to MBP. TheseHSP60-specific T-cells secreted low amounts of both IL-10 (22±5 pg/ml)and IFNγ (80±15 pg/ml) upon stimulation in vitro with HSP60 (data notshown). No cytokine release was detected when splenocytes frompcDNA3-treated animals were stimulated with HSP60. No significantdifferences were seen between the different experimental groups neitherin T-cell proliferation nor in cytokine-release in response tostimulation with Con A (data not shown). Thus pHSP60 vaccination induceda low, but specific T-cell response to HSP60; the immune responseelicited by pHsp60 vaccination is capable of affecting the immunereactions that characterize AA.

Example 11 Cytokine Secretion in AA Rats Vaccinated with pHSP60 pHSP65or Hu3/IFA

Twenty-six days after the induction of AA, LNC were prepared fromuntreated rats, or animals that had been treated with pcDNA3 or PBS/IFA.LNC were stimulated in vitro with a collective of antigens previouslyknown to be targeted or associated with AA: HSP60, HSP65, PPD, P176-90(which contains the 180-188 epitope of HSP65 (3)) and Hu3 describedhere. Hu12 and OVA were included as control antigens. The results wereessentially the same whether the AA was induced in untreated rats, or inrats pre-treated with injections of PBS/IFA or pcDNA3. FIG. 11 depictsthe results obtained with LNC isolated from pcDNA3-treated animals,showing the cytokines released to the culture medium. LNC frompCDNA3-treated animals showed a strong proliferative response to PPD,and low but significant responses to HSP65 and P176-90, while noproliferation was detected after stimulation with HSP60, Hu3 or Hu12.Although the proliferative response to P176-90 was quite low,stimulation with this peptide led to the release of IFNγ to at least thesame levels as those achieved by stimulation with PPD. IFNγ was secretedto a lower extent in response to HSP65, while no secretion was detectedupon stimulation with HSP60, Hu3 or Hu12. IL-10 and TGFβ1 were detectedonly upon activation with PPD. Thus, induction of AA activates T-cellsthat almost exclusively secrete IFNγ in response to activation withmycobacterial antigens, and that do not appear to recognize HSP60 or itspeptides Hu3 and Hu12.

FIG. 12A depicts the proliferative response and cytokine secretion ofcells isolated from animals treated with pHSP60, pHSP65 or Hu3/IFAexpressed as the percent change in reactivity relative to theproliferation seen using cells from their respective controls (pcDNA3 orPBS/IFA-treated animals). Animals protected from AA showed increasedproliferative responses to mycobacterial antigens (PPD, HSP65 andP176-90). The increase in the proliferation to HSP65 and PPD wasstronger in pHSP60-treated animals. Also the response to mammalian HSP60was up-regulated throughout all the groups, but this effect was moremarked in pHSP60 vaccinated animals. Moreover, at day 26 after AAinduction, the response to HSP60 was significantly higher than thatdetected at the end of the immunization protocol (Stimulation Index,SI=2.4±0.39 vs. SI=4.44±0.37 respectively, p<0.05). In addition, Hu3 wasonly recognized by animals immunized with pHSP60, or with Hu3 itself,while there were no responses directed towards Hu12. Cells fromHU12/IFA-treated rats proliferated upon stimulation with HU12 (data notshown). None of the experimental groups showed significant responses toOVA, and they did not differ in their response to Con A (data notshown). Thus, modulation of AA by vaccination with pHSP60, pHSP65 or Hu3is accompanied by the up-regulation of T-cell proliferative responses toboth self- and mycobacterial antigens. The phenotype of these augmentedcellular responses has been characterized in terms of cytokine releaseprofiles.

As shown in FIG. 12B, Secretion of IFNγ was increased in LNC from: A,pHSP60-treated rats stimulated with HSP60, Hu3 or Hu12; B,pHSP65-treated rats stimulated with HSP60 and C, in Hu3-treated ratsstimulated with Hu3. Thus, modulation of AA is associated with areduction in IFNγ release to P176-90 and an increase in the release ofIFNγ to HSP60. As shown in FIG. 12C, The study of IL-10 secretionrevealed that LNC from animals protected from AA (either by DNA orpeptide vaccination) showed increased secretion of IL-10 in response toin vitro stimulation with mycobacterial antigens. In cells from all thegroups protected we found secretion of IL-10 in response to stimulationwith PPD, HSP65 (pHSP60 and pHSP65-treated animals) or P176-90 (pHSP60-and Hu3-treated rats). Release of IL-10 upon stimulation with HSP60 andits peptides was not uniform, and only found in cells from pHSP60 orpHSP65-vaccinated animals. Therefore protection was associated with theinduction of IL-10 secretors responsive to mycobacterial antigens (PPDor P180-90) and, in DNA vaccinated animals, to mammalian HSP60. Theanalysis of TGFβ1 release from LNC taken from all the groups protectedfrom AA, (either by DNA or peptide vaccination), showed an increase inthe secretion of TGFβ1 in response to stimulation with mycobacterialantigens and HSP60 or its peptides (FIG. 12D). However, this effect wasstronger in DNA-treated rats.

All together, the results suggest that modulation of AA by treatmentwith specific DNA or peptide is associated with three observations. A,decreased secretion of IFNγ upon stimulation with the HSP65 peptideP178-190. B, the induction HSP60-specific T-cells that secrete IFNγ. C,the appearance of IL-10 and TGFβ1 secretors specific for mycobacterialantigens and HSP60.

Example 12 Inhibition of AA by DNA Vaccines Encoding Human HSP70 andHuman HSP90

The full-length cDNA of human HSP70 (pHSP70) or human HSP90 (pHSP90)were cloned into the pcDNA3 vector. The gene constructs were found to befunctional in an in vitro transcription/translation system (data notshown). Rats were immunized with pHSP90 and pHSP70 following the samescheme of vaccination used for pHSP60, and 12 days after the lastinjection of DNA, AA was induced. FIG. 13A shows that in rats vaccinatedwith pHSP70 or pHSP90 there was a significant inhibition of AA.Inhibition of AA was also seen as a reduction in the maximal score (FIG.13B), leg swelling (FIG. 13C) and a significant delay in the mean day ofdisease onset (FIG. 13D).

In order to gain some insight into the mechanism that mediatesprevention of AA by treatment with pHSP70 or pHSP90, the induction ofantibodies of the IgG isotype to the antigen encoded by the vector wasstudied. FIGS. 14A and 14B depict the results obtained for pHSP70- andpHSP90-treated rats respectively. Vaccination with the DNA constructsinduced specific antibodies, indicating that both constructs areimmunogenic; and the humoral response induced is up-regulated uponinduction of AA.

Example 13 Cytokine Secretion and In Vitro Proliferation of LNC Isolatedfrom AA Rats Vaccinated with pHSP70 or pHSP90

FIGS. 15A and 15B shows that draining lymph node DLN cells fromimmunized animals showed a dose response proliferation upon activationwith the protein encoded by the immunizing vector. Furthermore, theanalysis of the cytokines released in response to antigen-specificstimulation revealed that cells taken from pHSP90-immunized animalssecreted both IL-10 and IFNγ in response to activation with HSP90 (FIGS.16A and 16B). There was no IFNγ or IL-10 secretion when cells frompHSP70-treated animals were stimulated with HSP70 (data not shown).

The immune response to a panel of antigens in DLN cells isolated frompcDNA3-, pHSP70- or pHSP90-treated rats was detected 26 days after theinduction of AA. The results are shown in FIG. 17 as the percentage ofthe proliferation observed in untreated rats. Vaccination with pHSP70 orpHSP90 led to a significant up-regulation in the proliferative responseto HSP65, HSP71 and PPD, and this increase in the immune response wasstronger in pHSP70 vaccinated rats. To further characterize the natureof the up-regulated immune response in pHSP70- and pHSP90-vaccinated,cytokine secretion in response to in vitro stimulation with the sameAA-related antigens was detected. IFNγ secretion in response tostimulation with HSP65, its epitope P176-188 or PPD was down-regulatedin animals treated with pHSP70 or pHSP90 (FIG. 18A). In addition, IL-10secretion was up-regulated upon in vitro activation with PPD, HSP71,HSP65 or HSP60, both in pHSP70- and pHSP90-vaccinated animals (FIG.18B). Finally, TGFβ1 release upon stimulation with HSP65 and its peptideP176-80 was increased in cells taken from pHSP90-vaccinated animals, andthere was a slight but significant release of in response to stimulationwith PPD in cells from pHSP70-treated animals (FIG. 18C). Note that thedecrease in IFNγ secretion and the concomitant increase in IL-10 andTGFβ1 release was stronger in the group treated with pHSP90; in thisgroup AA was inhibited even more effectively than in pHSP70-immunizedrats. TABLE I Overlapping peptides of human HSP60, region 1-275 PeptidePosition Sequence Hu1    1-20 MLRLPTVFRQMRPVSRVLAP Hu2   16-35RVLAPHLTRAYAKDVKFGAD Hu3   31-50 (SEQ ID NO:3) KFGADARALMLQGVDLLADA Hu4  46-65 LLADAVAVTMGKGRTVIIE Hu5   61-80 TVIIEQSWGSPKVTKDGVTV Hu6   76-95DGVTVAKSIDLKDKYKNIGA Hu7   91-110 KNIGAKLVQDVANNTNEEAG Hu8  106-125NEEAGKGTTTATVLARSIAK Hu9  121-140 RSIAKEGFEKISKGANPVEI Hu10 136-155NPVEIRRGVMLAVDAVIAEL Hu11 151-170 VIAELKKQSKPVTTPEEIAQ Hu12 166-185EEIAQVATISANGDKEIGNI Hu13 181-199 EIGNIISDAMKKVGRKGVI Hu14 195-214RKGVITVKDGKTLNDELEII Hu15 210-229 ELEIIEGMKFDRGYISPYFI Hu16 225-244SPYFINTSKGQKCEFQDAYV Hu17 240-259 QDAYVLLSEKKISSIQSIVP Hu18 255-275QSIVPALEIANAHRKPLVIIA

TABLE II Plasmids constructed containing overlapping fragments of thehsp60 gene Plasmid Position Corresponding Peptides included pI     1-140Hu1-Hu9 (a.k.a. P1-P9; SEQ ID NO:1) pII  130-260 Hu10-Hu18 (a.k.a.P10-P18; SEQ ID NO:2) pIII 250-410 NS pIV  400-470 NS pIV  460-540 NSThe position is expressed as amino acid residue numbers.NS = Not synthesized as individual peptides.

TABLE III Comparison of human HSP60, rat HSP60 and mycobacterial HSP65in the region corresponding to the Hu3 sequence^(a,b). H. sapiens 31KFGADARALMLQGVDLLADA 50 R. norvergicus 31 KFGADARALMLQGVDLLADA 50 M.tuberculosis 5 AYDEE ARRGLERG LNALADA 24^(a) H. sapiens, Homo sapiens; R. norvergicus, Rattus norvergicus; M.tuberculosis, Mycobacterium tuberculosis.^(b)Residues sharing identity with the corresponding sequence of humanHSP60 are shown in bold, and conserved substitutions are shown asunderlined residues.References:

-   1. Wauben, M. H. M., J. P. A. Wagenaar-Hilbers, and W. van    Eden. 1994. Adjuvant Arthritis. In Autoimmune Disease Models. I. R.    Cohen, and A. Miller, eds. Academic Press, Inc., California, USA.-   2. van Eden, W., J. E. Thole, R. van der Zee, A. Noordzij, J. D. van    Embden, E. J. Hensen, and I. R. Cohen. 1988. Cloning of the    mycobacterial epitope recognized by T lymphocytes in adjuvant    arthritis. Nature 331:171.-   3. van Eden, W., J. Holoshitz, Z. Nevo, A. Frenkel, A. Klajman,    and I. R. Cohen. 1985. Arthritis induced by a T-lymphocyte clone    that responds to Mycobacterium tuberculosis and to cartilage    proteoglycans. Proc Natl Acad Sci USA 82:5117.-   4. Holoshitz, J., A. Matitiau, and I. R. Cohen. 1984. Arthritis    induced in rats by cloned T lymphocytes responsive to mycobacteria    but not to collagen type II. J Clin Invest 73:211.-   5. Holoshitz, J., Y. Naparstek, A. Ben-Nun, and I. R. Cohen. 1983.    Lines of T lymphocytes induce or vaccinate against autoimmune    arthritis. Science 219:56.-   6. Billingham, M. E., S. Carney, R. Butler, and M. J. Colston. 1990.    A mycobacterial 65-kD heat shock protein induces antigen-specific    suppression of adjuvant arthritis, but is not itself arthritogenic.    J Exp Med 171:339.-   7. Hogervorst, E. J., L. Schouls, J. P. Wagenaar, C. J. Boog, W. J.    Spaan, J. D. van Embden, and W. van Eden. 1991. Modulation of    experimental autoimmunity: treatment of adjuvant arthritis by    immunization with a recombinant vaccinia virus. Infect Immun    59:2029.-   8. Ragno, S., M. J. Colston, D. B. Lowrie, V. R. Winrow, D. R.    Blake, and R. Tascon. 1997. Protection of rats from adjuvant    arthritis by immunization with naked DNA encoding for mycobacterial    heat shock protein 65. Arthritis Rheum 40:277.-   9. Moudgil, K. D., T. T. Chang, H. Eradat, A. M. Chen, R. S.    Gupta, E. Brahn, and E. E. Sercarz. 1997. Diversification of T cell    responses to carboxy-terminal determinants within the 65-kD    heat-shock protein is involved in regulation of autoimmune    arthritis. J Exp Med 185:1307.-   10. Anderton, S. M., R. van der Zee, B. Prakken, A. Noordzij, and W.    van Eden. 1995. Activation of T cells recognizing self 60-kD heat    shock protein can protect against experimental arthritis. J Exp Med    181:943.-   11. Yang, X. D., J. Gasser, and U. Feige. 1992. Prevention of    adjuvant arthritis in rats by a nonapeptide from the 65-kD    mycobacterial heat shock protein: specificity and mechanism. Clin    Exp Immunol 87:99.-   12. van Eden, W., U. Wendling, L. Paul, B. Prakken, P. van Kooten,    and R. van der Zee. 2000. Arthritis protective regulatory potential    of self-heat shock protein cross-reactive T cells. Cell Stress    Chaperones 5:452.-   13. Lopez-Guerrero, J. A., J. P. Lopez-Bote, M. A. Ortiz, R. S.    Gupta, E. Paez, and C. Bernabeu. 1993. Modulation of adjuvant    arthritis in Lewis rats by recombinant vaccinia virus expressing the    human 60-kilodalton heat shock protein. Infect Immun 61:4225.-   14. Lopez-Guerrero, J. A., M. A. Ortiz, E. Paez, C. Bernabeu,    and J. P. Lopez-Bote. 1994. Therapeutic effect of recombinant    vaccinia virus expressing the 60-kd heat-shock protein on adjuvant    arthritis. Arthritis Rheum 37:1462.-   15. Quintana, F. J., P. Carmi, F. Mor, and I. R. Cohen. 2002.    Inhibition of adjuvant arthritis by a DNA vaccine encoding human    heat shock protein 60. J Immunol 169:3422.-   16. Anderton, S. M., R. van der Zee, A. Noordzij, and W. van    Eden. 1994. Differential mycobacterial 65-kDa heat shock protein T    cell epitope recognition after adjuvant arthritis-inducing or    protective immunization protocols. J Immunol 152:3656.-   17. Quintana, F. J., A. Rotem, P. Carmi, and I. R. Cohen. 2000.    Vaccination with empty plasmid DNA or CpG oligonucleotide inhibits    diabetes in nonobese diabetic mice: modulation of spontaneous 60-kDa    heat shock protein autoimmunity. J Immunol 165:6148.

1. A DNA vaccine for treating a T cell-mediated inflammatory autoimmunedisease comprising a recombinant construct, the recombinant constructcomprising a nucleic acid sequence encoding a mammalian heat shockprotein or an active fragment thereof, the nucleic acid sequence beingoperatively linked to one or more transcription control sequences in asuitable expression system, enabling in vivo expression of the encodedprotein or the active fragment thereof in a human host.
 2. The DNAvaccine of claim 1, wherein the mammalian heat shock protein is a humanheat shock protein.
 3. The DNA vaccine of claim 1, wherein the heatshock protein is heat shock protein 60 (HSP60), heat shock protein 70(HSP70) or heat shock protein 90 (HSP90).
 4. The DNA vaccine of claim 3,wherein the recombinant construct comprises a nucleic acid sequenceencoding a fragment of HSP60, HSP70 or HSP90.
 5. The DNA vaccine ofclaim 4, wherein the nucleic acid sequence encodes a fragment of HSP60,with the fragment having an amino acid sequence selected from the groupconsisting of amino acids 1-140 of HSP60 (denoted as SEQ ID NO: 1),amino acids 130-260 of HSP60 (denoted as SEQ ID NO: 2), and amino acids31-50 of HSP60 (denoted as SEQ ID NO: 3).
 6. The DNA vaccine of claim 1,wherein the T cell-mediated inflammatory autoimmune disease isrheumatoid arthritis, collagen II arthritis, multiple sclerosis,autoimmune neuritis, systemic lupus erythematosus, psoriasis, juvenileonset diabetes, Sjogren's disease, thyroid disease, sarcoidosis,autoimmune uveitis, inflammatory bowel disease (Crohn's and ulcerativecolitis) or autoimmune hepatitis.
 7. The DNA vaccine of claim 1, whereinthe transcription control sequences are selected from the groupconsisting of RSV control sequences, CMV control sequences, retroviralLTR sequences, SV-40 control sequences and (3-ACTIN? control sequences.8. The DNA vaccine of claim 1, wherein the recombinant construct isincorporated into an eukaryotic expression vector.
 9. The DNA vaccine ofclaim 8, wherein the eukaryotic expression vector is selected from thegroup consisting of PCDNA3, pcDNA3.1 (+/−), pZeoSV2 (+/−), pSecTag2,pDisplay, pEF/myc/cyto, PCMV/MYC/CYTO, pCR3.1, pCI, PBK-RSV, PBK-CMV andPTRES.
 10. A recombinant construct comprising a nucleic acid sequenceencoding a fragment of mammalian HSP60, the fragment having an aminoacid sequence selected from the group consisting of amino acids 1-140 ofHSP60 (denoted as SEQ ID NO: 1), amino acids 130-260 of HSP60 (denotedas SEQ ID NO: 2), and amino acids 31-50 of HSP60 (denoted as SEQ ID NO:3), with the nucleic acid sequence being operatively linked to one ormore transcription control sequences.
 11. The construct of claim 10,wherein the mammalian HSP60 is human HSP60.
 12. The construct of claim10, wherein the transcription control sequences are selected from thegroup consisting of RSV control sequences, CMV control sequences,retroviral LTR sequences, SV-40 control sequences and P-ACTIN controlsequences.
 13. The construct of claim 10, wherein the recombinantconstruct is incorporated into an eukaryotic expression vector.
 14. Theconstruct of claim 13, wherein the eukaryotic expression vector isselected from the group consisting of: PCDNA3, pcDNA3.1 (+/−), pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pCI,PBK-RSV, PBK-CMV and PTRES.
 15. A pharmaceutical composition fortreating a T cell-mediated autoimmune disease comprising (a) arecombinant construct comprising an isolated nucleic acid sequenceencoding a heat shock protein or an active fragment thereof, with thenucleic acid sequence being operatively linked to one or moretranscription control sequences; and (b) a pharmaceutically acceptablecarrier.
 16. The pharmaceutical composition of claim 15, which is usefulfor a T cell mediated inflammatory autoimmune disease other than insulindependent diabetes mellitus (IDDM).
 17. The pharmaceutical compositionof claim 16, wherein the T cell-mediated inflammatory autoimmune diseaseis rheumatoid arthritis, collagen II arthritis, multiple sclerosis,autoimmune neuritis, systemic lupus erythematosus, psoriasis, juvenileonset diabetes, Sjogren's disease, thyroid disease, sarcoidosis,autoimmune uveitis, inflammatory bowel disease (Crohn's and ulcerativecolitis) or autoimmune hepatitis.
 18. The pharmaceutical composition ofclaim 15, wherein the heat shock protein is HSP60, HSP70 or HSP90, or anactive fragment thereof.
 19. The pharmaceutical composition of claim 18,wherein the active fragment has an amino acid sequence selected from thegroup consisting of amino acids 1-140 of HSP60 (denoted as SEQ ID NO:1), amino acids 130-260 of HSP60 (denoted as SEQ ID NO: 2), and aminoacids 31-50 of HSP60 (denoted as SEQ ID NO: 3).
 20. The pharmaceuticalcomposition of claim 15, wherein the carrier comprises a deliveryvehicle that delivers the nucleic acid sequences to an individual. 21.The pharmaceutical composition of claim 20, wherein the delivery vehicleis selected from the group consisting of liposomes, micelles, emulsionsand cells.
 22. The pharmaceutical composition of claim 15, wherein thetranscription control sequences are selected from the group consistingof RSV control sequences, CMV control sequences, retroviral LTRsequences, SV-40 control sequences and P-ACTIN control sequences. 23.The pharmaceutical composition of claim 15, wherein the recombinantconstruct is incorporated into an eukaryotic expression vector.
 24. Thepharmaceutical composition of claim 23, wherein the eukaryoticexpression vector is selected from the group consisting of pcDNA3,pcDNA3.1 (+/−), pZeoSV2 (+/−), pSecTag2, pDisplay, pEF/myc/cyto,PCMV/MYC/CYTO, pCR3.1, pCI, PBK-RSV, PBK-CMV and PTRES.
 25. A method oftreating a T cell-mediated inflammatory autoimmune disease, whichcomprises administering to an individual in need thereof a therapeuticcomposition comprising a recombinant construct that includes an isolatednucleic acid sequence encoding a heat shock protein or an activefragment thereof, with the nucleic acid sequence being operativelylinked to one or more transcription control sequences, thereby treatingthe disease.
 26. The method of claim 25, wherein the heat shock proteinis HSP60, HSP70, HSP90, or an active fragment thereof.
 27. The method ofclaim 26, wherein the active fragment having amino acid sequenceselected from the group consisting of amino acids 1-140 of HSP60(denoted as SEQ ID NO: 1), amino acids 130-260 of HSP60 (denoted as SEQID NO: 2), and amino acids 31-50 of HSP60 (denoted as SEQ ID NO: 3). 28.The method of claim 25, wherein the T cell-mediated inflammatoryautoimmune disease is rheumatoid arthritis, collagen II arthritis,multiple sclerosis, autoimmune neuritis, systemic lupus erythematosus,psoriasis, juvenile onset diabetes, Sjogren's disease, thyroid disease,sarcoidosis, autoimmune uveitis, inflammatory bowel disease (CROLM&#x0;Sand ulcerative colitis) or autoimmune hepatitis.
 29. The method of claim25, wherein the individual to be treated is selected from the groupconsisting of humans and non-human mammals.
 30. The method of claim 25,wherein the therapeutic composition is administered to the individualprior to the appearance of disease symptoms.
 31. The method of claim 25,wherein the therapeutic composition is administered by intravenousinjection, intramuscular injection, or by aerosol, oral, percutaneous ortopical administration.
 32. A method for treating a T cell-mediatedinflammatory autoimmune disease which comprises the steps of (a)obtaining cells from an individual; (b) transfecting the cells in vitrowith a recombinant construct comprising an isolated nucleic acidsequence encoding a heat shock protein or an active fragment thereof,said nucleic acid sequence being operatively linked to one or moretranscription control sequences; and (c) reintroducing the transfectedcells to the individual, thereby treating the disease.
 33. The method ofclaim 32, wherein the heat shock protein is HSP60, HSP70, HSP90, or anactive fragment thereof.
 34. The method of claim 33, wherein the activefragment has an amino acid sequence selected from the group consistingof amino acids 1-140 of HSP60 (denoted as SEQ ID NO: 1), amino acids130-260 of HSP60 (denoted as SEQ ID NO: 2), and amino acids 31-50 ofHSP60 (denoted as SEQ ID NO: 3).
 35. The method of claim 32, wherein theT-cell mediated inflammatory autoimmune disease is rheumatoid arthritis,collagen II arthritis, multiple sclerosis, autoimmune neuritis, systemiclupus erythematosus, psoriasis, juvenile onset diabetes, Sjogren'sdisease, thyroid disease, sarcoidosis, autoimmune uveitis, inflammatorybowel disease (Crohn's and ulcerative colitis) or autoimmune hepatitis.36. The method of claim 32, wherein the individual to be treated isselected from the group consisting of humans and non-human mammals. 37.A method of treating arthritis, which comprises administering to anindividual in need thereof a therapeutic composition comprising (a) afragment of mammalian HSP60 having amino acid sequence corresponding toamino acids 31-50 of HSP60 (denoted as SEQ ID NO: 3); and (b) apharmaceutically acceptable carrier, thereby treating arthritis.
 38. Themethod of claim 37, wherein the carrier comprises a delivery vehiclethat delivers the fragment to the individual.
 39. The method of claim38, wherein the delivery vehicle is selected from the group consistingof liposomes, micelles, emulsions and cells.
 40. A method of treatingarthritis which comprises the steps of (a) obtaining cells from anindividual; (b) exposing the cells in vitro with an active amount of afragment of mammalian HSP60 having amino acid sequence corresponding toamino acids 31-50 of HSP60 (denoted as SEQ ID NO: 3); and (c)reintroducing the exposed cells to the individual, thereby treatingarthritis.
 41. The method of claim 40, wherein the cells are autologousT cells.
 42. The method of claim 40, wherein the mammalian HSP60 ishuman HSP60.